Shear separation method and system

ABSTRACT

A shear separation system or method uses shear lift forces and permeate drag forces to separate substances having a size less than a predetermined separation size from substances having a size greater than the separation size. The balance of the drag forces and lift forces affects a predetermined separation size in that the balance of forces retards the transmembrane passage of substances larger than the separation size yet allows substances smaller than the separation size to pass through the permeable membrane. The shear separation device, method and system can be employed to separate or concentrate from a process fluid substances larger or smaller than the predetermined separation size

FIELD OF THE INVENTION

[0001] The present invention relates to shear separation methods andsystems and, more particularly, to shear separation methods and systemswherein microfiltration, ultrafiltration, diafiltration, orconcentration can be achieved.

BACKGROUND OF THE INVENTION

[0002] Separation methods and systems, such as those employing filters,typically are employed to separate one or more components or substancesof a fluid from other components or substances in the fluid. As usedherein, the term “fluid” includes liquids, gases, and mixtures andcombinations of liquids, gases and/or solids. Conventional separationprocesses include a wide variety of common processes, such as classic orparticle filtration, microfiltration, ultrafiltration, nanofiltration,reverse osmosis (hyperfiltration), dialysis, electrodialysis,prevaporation, water splitting, sieving, affinity separation,purification, affinity purification, affinity sorption, chromatography,gel filtration, bacteriological filtration, and coalescence. Typicalseparation devices and systems may include dead end filters, cross-flowfilters, dynamic filters, vibratory separation systems, disposablefilters, regenerable filters including backwashable, blowback andsolvent cleanable, and hybrid filters which comprise different aspectsof the various above described devices.

[0003] Accordingly, as used herein, the term “separation” shall beunderstood to include all processes, including filtration, wherein oneor more components of a fluid is or are separated from the othercomponents of the fluid. The terms “filter”, “separation medium”, and“permeable membrane” shall be understood to include any medium made ofany material that allows one or more substances in a fluid to passtherethrough in order to separate those substances from the othercomponents of the fluid. The terminology utilized to define the varioussubstances in the fluid undergoing separation and the products of theseprocesses may vary widely depending upon the application, e.g., liquidor gas filtration, and the type of separation system utilized, e.g.,dead end or open end systems; however, for clarity, the following termsshall be utilized. The fluid which is input to the separation systemshall be referred to as process fluid and construed to include any fluidundergoing separation. The portion of the fluid which passes through theseparation medium shall be referred to as permeate and construed toinclude filtrate as well as other terms. The portion of the fluid whichdoes not pass through the separation medium shall be referred to asretentate and construed to include concentrate, bleed fluid, as well asother terms.

[0004] While many separation applications are quite routine, theseparation of relatively small particles or substances from fluidsrequires separation protocols able to achieve a precise separation size(resolution) with minimal fouling (e.g., clogging with the smallparticles). This is particularly the situation when separating proteins(natural or recombinant) and other components from process fluids suchas milk or products derived from milk (e.g., skim milk, whey, etc.).

[0005] Milk contains, among other things, fats, proteins (casein and avariety of other proteins such as β-lactoglobulin, α-lactalbumin, serumalbumin, and immunoglobulins), salts, sugar (lactose), and variousvitamins (such as vitamins A, C, and D, along with some B vitamins) andminerals (primarily calcium and phosphorus). The composition of milkvaries with the species, breed, feed, and condition of the animal fromwhich the milk is obtained. Moreover, a wide variety of milk or wheyproteins are employed as functional and nutritional ingredients inbakery products, pasta, confections, beverages, meats, and other foodproducts. In addition, milk has proven a valuable source of biologicallyor medically important products. For example, it is possible to obtainantibodies by vaccinating lactating animals and collecting antibodiesfrom their milk (see, e.g., U.S. Pat. Nos. 5,260,057 (Corcle et al.) and3,128,230 (Heinbach et al.)). Moreover, many species of animals havebeen genetically engineered to express recombinant proteins in milk.See, e.g., Gordon et al., Biotechnology, 5(11), 1183-87 (1987) (mice);Ebert et al., Biotechnology, 12(7), 699-702 (1994) (goats); Lee et al.,J. Control. Release, 29(3), 213-21 (1994) (dairy cows); Limonta et al.,J. Biotechnol., 40(1), 49-58 (1995) (rabbits); Clark et al.,Biotechnology, 7(5), 487-92 (1989) (sheep). Examples of such recombinantproteins are peptide hormones (e.g., growth hormones (Archer et al.,Proc. Nat. Acad. Sci. USA, 91(15), 6840-44 (1994)), tissue plasminogenactivator (tPA) (Ebert et al., supra), etc.), blood coagulation factorsor subunits of them (e.g., factors VIII and IX (Clark et al., supra)),anticoagulation factors or subunits of them (e.g., anti-thrombin III andhuman protein C), other blood proteins (e.g., serum albumin (Barash etal., Mol. Repro. Dev., 45(4), 421-30 (1996)), beta-globin,α1-antitrypsin (Archibald et al, Proc. Nat. Acad. Sci. USA, 87(13),5178-82 (1990)), proteins for foodstuffs, enzymes, and other proteins(e.g., collagen, cystic fibrosis transmembrane conductance regulator(CFTR), antibodies, etc.). See, e.g., U.S. Pat. No. 4,873,316 (Meade etal.), U.S. Pat. No. 5,589,604 (Drohan et al.), and U.S. Pat. No.5,476,995 (Clark et al.). Secretion of recombinant proteins into themilk of transgenic animals is an efficient method of producing suchproteins; concentrations approaching 10 g/l have been reported.

[0006] Commercially produced milk commonly undergoes pasteurization tomitigate bacterial growth and homogenization to improve fat dispersionstability. Moreover, in the commercial processing of milk products, itis desirable in certain instances to remove as much fat as possible fromthe milk products.

[0007] Conventional milk processing heretofore has involved the use ofmechanical separation (centrifugation), evaporation/crystallization,steam injection, electrodialysis, reverse osmosis, ultrafiltration, gelfiltration, diafiltration, and/or ion exchange chromatography. Forexample, whey typically is subjected to mechanical separation (e.g.,centrifuged) to remove fat, condensed via evaporation to increase solidscontent, and then spray dried or used for lactose crystallization. Afterdesludging, the residual concentrate is dried, which yields whey powdercontaining about 11-14% protein (which usually is denatured,particularly during the evaporation/condensation step). The whey powdercan be subjected to electrodialysis to remove ash and thereby preparedemineralized whey powder. Alternatively, the whey powder can besubjected to reverse osmosis to remove water, thereby obtaining wheypowder containing about 12-15% protein. Such a whey powder can besubjected to ultrafiltration or gel filtration to remove further ash andlactose and thereby obtain a whey protein concentrate containing about30-50% protein, which, in turn, can be subjected to diafiltration or ionexchange chromatography to remove yet more ash and lactose so as toobtain whey protein concentrates containing about 50-90% protein.

[0008] Such conventional processing methods carry with them manydisadvantages, such as long processing times, high costs, and poor orinconsistent component fractionation. Moreover, it is often difficult toseparate a recombinant protein from fluids such as milk by these methodswithout denaturing or damaging the protein, and it is also difficult toseparate different proteins and particles of interest within milk orother fluids. Many of these difficulties are attributable to theaforementioned problems attendant with separating relatively smallparticles from fluids, namely poor resolution and filter fouling.

[0009] One advancement greatly reducing filter fouling is to employseparation methods and systems generating a shear layer at the surfaceof a filter. A layer of fluid which is adjacent to the surface of afilter and which is in a state of rapid shear flow parallel to thesurface of the filter tends to minimize fouling of the filter bysweeping contaminant matter in the process fluid from the filter.Generally two such technologies can be used for developing a shearlayer: cross flow and dynamic filtration. In cross flow systems, highvolumes of fluid are driven through passages bounded by the filtersurface and possibly the inner surface of the filter housing, therebycreating the necessary shear. Simply stated, process fluid is pumpedacross the upstream surface of the filter at a velocity high enough todisrupt and back mix the boundary layer. In dynamic filter systems, thenecessary shear is created by motion of one or more surfaces that can beprovided for that purpose (e.g., the filter surface, the filter vessel,or any contained discs, impellers, etc.). Two widely used configurationsare cylinder devices and disc devices. Unlike cross flow filtrationsystems, the shear created in dynamic filtration systems at the fluidinterface is substantially or nearly independent of any cross flow fluidvelocity. Traditional cross-flow filtration systems generate sheargenerally between about 5,000 sec⁻¹ and about 10,000 sec⁻¹, while shearsgenerated by dynamic filtration systems are between about 100,000 sec⁻¹and about 500,000 sec⁻¹.

[0010] While dynamic and traditional cross-flow filtration systems canachieve reduced fouling, the size or molecular weight cutoff of theparticles of interest is controlled by the separation medium. In bothsystems, the actual separation or filtering action is effected by theseparation medium, the pores of which are sized to remove or separatethe particles of interest. Particles larger than the pores are unable topass through the separation medium while particles smaller than thepores readily pass through the medium. Due to the foulingcharacteristics of a process fluid and the inherent difficulties inengineering filter media with uniform and predefined pore sizes, highresolution separation of relatively small particles (e.g., molecularweight particles) has been exceedingly difficult using dynamic and crossflow filtration systems.

[0011] To address these drawbacks, there is a need for improved meansfor separating small substances from a solution or a suspension, even ahighly fouling solution or suspension. In particular, there is a needfor means of effectively concentrating particles of a given molecularweight (e.g., specific proteins), thereby achieving fractionation ofsuch solutions or suspensions. The present invention provides a reliableand efficient means for the separation of small particles or substances(e.g., molecular size particles) from a variety of fluids (e.g.,solutions, suspensions, emulsions, etc.), especially milk products.

SUMMARY OF THE INVENTION

[0012] The present invention provides shear separation systems andmethods for treating a process fluid to separate substances having asize less than a predetermined separation size from substances having asize greater than the separation size. Permeate flow through a permeablemembrane establishes a drag force acting on substances upstream of thepermeable membrane, and a shear rate is created at the surface of thepermeable membrane to establish a lift force acting on substancesupstream of the permeable membrane. The balance of the drag force andlift force effects the predetermined separation size in that the balanceof forces retards the transmembrane passage of substances larger thanthe separation size yet allows substances smaller than the separationsize to pass through the permeable membrane. Shear separation systemsand methods embodying the invention can be employed to separate orconcentrate from a process fluid substances larger or smaller than thepredetermined separation size by collecting them from the retentate orpermeate.

[0013] The present invention effects the separation or concentration ofrelatively small particles or substances, such as proteins and otherbiological molecules, far more effectively and with much greaterflexibility than conventional systems or methods. Therefore, theseparation methods and systems of the present invention are useful forthe efficient separation of substances from a wide variety of fluids.For example, the present invention can treat milk products to reduce thebacteria and fat therein and/or to recover proteins therefrom, evenfractionating such fluids. These and other advantages of the presentinvention, as well as additional inventive features, will be apparentfrom the drawings and the detailed description outlined below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a diagrammatic representation of a feed streamtangential velocity profile across a permeable membrane and a permeatevelocity profile emerging from the permeable membrane.

[0015] FIGS. 2A-2C are diagrammatic representations of the effect ofturbulence fluctuations on the feed stream tangential velocity profileacross a permeable membrane and the permeate velocity profile emergingfrom the permeable membrane.

[0016]FIG. 2A depicts fluctuating velocity in each of three fluid flowregions.

[0017]FIG. 2B depicts the convection of velocity fluctuations towardsthe upstream surface of a permeable membrane.

[0018]FIG. 2C depicts the relative increase in the thickness of theviscous sublayer if the surface of the permeable membrane is not smooth.

[0019]FIG. 3 is a diagrammatic representation of the forces acting on aparticle suspended in process fluid subjected to the feed streamtangential flow across a permeable membrane and the permeate flowemerging from the permeable membrane.

[0020]FIG. 4 is a diagrammatic representation of the effect of theviscous sublayer on large particles.

[0021]FIG. 5 is a diagrammatic representation of a shear separationsystem including a controller for controlling one or both of the shearlift and permeate drag forces.

[0022]FIGS. 6A and 6B are graphical representations of the experimentalresults of protein separation using a shear separation method of thepresent invention in conjunction with 300 kD (FIG. 6A) and 70 kD (FIG.6B) membranes.

DETAILED DESCRIPTION

[0023] The present invention provides shear separation systems andmethods of treating a process fluid to separate substances having a sizeless than a predetermined separation size from substances having a sizegreater than the separation size by using the shear lift and permeatedrag forces which are exerted on the substances near the surface of apermeable membrane to effect the separation. Consequently, the presentinvention is directed to shear separation systems and methods in whichhigh shear forces generated in a process fluid are used, not merely toprevent fouling of a permeable membrane, but also to separate, by size,substances in the process fluid.

[0024] The theory of operation described below is believed to be themechanism through which the shear separation systems, methods, anddevices of the present invention operate. However, other factors orphenomena not described can in some manner affect the shear separation.In addition, other theories of operation may be equally applicable toexplain the gross effect achieved by the shear separation systems,methods, and devices of the present invention.

[0025] Fluid flow in a direction tangential to a surface produces aregion of shear flow in proximity to the surface. The character of theshear flow depends upon the character of the surface and the velocity ofthe flow. A body or substance suspended in a shear flow near a surface,such as a permeable membrane, experiences a force, i.e., a shear liftforce, that tends to drive it away from the surface. The magnitude ofthis force depends on the character of the shear flow, the size of thesuspended body, and the distance of the body from the surface. Thesephenomena are well known and documented in the fluid mechanicsliterature. The characteristics of shear flows are described andanalyzed in, for example, Schlichting, H., Boundary Layer Theory. 7thed. New York: McGraw-Hill, 1979, and Tennekes, H., and Lumley, J. L., AFirst Course in Turbulence. Cambridge: MIT Press, 1972. Forces on a bodysuspended in a shear flow are described and analyzed in, for example,Goldman, A. J., Cox, R. G., Brenner, H. 1967. Slow Viscous Motion of aSphere Parallel to a Plane Wall-II Coette Flow, Chemical EngineeringScience 22, Saffman, P. G. 1965 The Lift on a Small Sphere in a SlowShear Flow, Journal of Fluid Mechanics, 22, part 2, and more recently,Otis, J. R., Altena, F. W., Mahar, J. T., and Belfort, G. 1986.Measurements of Single Spherical Particle Trajectories with LateralMigration in a Slit with One Porous Wall Under Laminar Flow Conditions,Experiments in Fluids, 4, and McLaughlin, John B. 1993, The Lift on aSmall Sphere in a Wall-Bounded Linear Shear Flows, Journal of FluidMechanics, 246.

[0026] The character of the shear flow depends on factors such as thecharacter of the surface of the permeable membrane and the velocity ofthe fluid flow across the permeable membrane. As the velocity of thefluid flow increases, the shear rate increases. However, higher fluidflow velocities result in more turbulent fluid flow, whereas lower fluidflow velocities produce more laminar flow. To effect the desired motionof the substances in the fluid, high shear is preferably used; hence thepresent inventive methods and systems preferably involve turbulent fluidflow. The shear rates are preferably from about 10,000 sec⁻¹ to about1,000,000 sec⁻¹ or more preferably from about 100,000 sec⁻¹ to about500,000 sec⁻¹, although even higher shears are suitable for use in theinventive methods and systems.

[0027]FIG. 1 illustrates a velocity profile that occurs near a permeablemembrane 10 in a tangential fluid flow, such as a flow of process fluidtangentially across the permeable membrane 10. The tangential fluidflow, indicated by arrows 12, is on the feed or upstream side of thepermeable membrane 10. A fraction of the fluid flow permeates throughthe permeable membrane 10 to the permeate or downstream side of thepermeable membrane 10. Although other situations are possible in variousembodiments of the present invention, pressure on each side of thepermeable membrane 10 is preferably uniform, with the greater pressurebeing on the upstream side, thereby contributing to the permeate flow.

[0028] When the tangential fluid flow is turbulent, three distinct flowregions are frequently discernible as illustrated in FIG. 1. Thedimensions of these regions vary with the shear rate. The region nearestthe upstream surface of the permeable membrane 10 is the viscoussublayer or, as it is often called, the laminar sublayer. The high shearrates preferably used in the present invention can produce viscoussublayers as thick as about 10 μm or more. In this region, turbulentfluctuations can be suppressed by the surface of the permeable membrane10, especially a smooth surface permeable membrane 10 as discussedherein. In the viscous sublayer, shear stress is transmitted through thefluid almost exclusively by viscosity, and velocity can increasesubstantially linearly with distance from the surface of the permeablemembrane 10.

[0029] The region farthest from the upstream surface of the permeablemembrane 10 is the turbulent region. In this region, the shear stress istransmitted almost exclusively by the random fluctuating motion of thefluid (i.e., Reynolds stress). In this turbulent region, the averagevelocity is substantially uniform and independent of the distance fromthe permeable membrane 10 as illustrated in FIG. 1.

[0030] The region intermediate the viscous sublayer and the turbulentregion is the inertial sublayer. The relatively high shear preferablyused in the present invention produces an inertial sublayer from about10 μm to about 500 μm thick. In this region, the shear stress can betransmitted partially by viscosity and partially by fluctuatingtransport of momentum. Viscosity dominates near the viscous sublayer,while fluctuating transport of momentum dominates near the turbulentregion. In this inertial sublayer, the average velocity generally variesin logarithmic relationship to the distance from the upper surface ofthe permeable membrane 10.

[0031] As illustrated in FIG. 1, the velocity of the fluid can have anaverage part which varies linearly near the upstream surface of thepermeable membrane 10, varies logarithmically farther away from thepermeable membrane 10, and finally becomes uniform still farther fromthe permeable membrane 10. Superimposed on this average velocity in eachregion is a fluctuating velocity. Relatively little of this fluctuatingmotion occurs in the viscous sublayer due to the surface effect of thepermeable membrane 10 discussed herein; more occurs in the inertialsublayer, and finally the motion of the fluid is dominated by thefluctuating velocity in the turbulent region. The fluctuating velocityin each region is illustrated in FIG. 2A by swirling arrows 16.

[0032] Because the membrane 10 is permeable, the permeate flow 12,illustrated in FIG. 1, convects velocity fluctuations 16 towards theupstream surface of the permeable membrane 10 as illustrated in FIG. 2B.If the permeate flow rate is high enough, the viscous sublayer can besubstantially eliminated. Accordingly, the permeate flow rate ispreferably controlled as explained herein so as to maximize the viscoussublayer.

[0033] Velocity fluctuations also can be increased in the viscoussublayer if the surface of the permeable membrane 10 is not smooth, asillustrated in FIG. 2C. Vortices can shed off of protrusions or regionsof recirculation and can contribute to velocity fluctuations anddiminish the role of the permeable membrane 10 in suppressingfluctuations. Accordingly, to maintain the integrity of the viscoussublayer, the upstream surface of the permeable membrane 10 preferablyis smooth (e.g., having a surface roughness which is small compared tothe thickness of the viscous sublayer).

[0034] A body or substance suspended in the process fluid near thesurface of the permeable membrane also experiences a force (i.e., thepermeate drag force) due to the permeate flow that tends to drive ittoward the surface. When the substance is a macromolecule, to generate alift force sufficient to balance the drag force of any useful permeateflow, the shear rate can be so large that turbulence becomes a dominantcharacteristic of the flow. However, as described herein, so long as thepermeate rate is not too large, the effect of velocity fluctuations inthe viscous sublayer is small, even if the turbulence is relativelystrong in the turbulent region. As a model for the operation of thepresent invention, it can be assumed, as illustrated in FIG. 3, that inthe viscous and inertial sublayers, the average velocity imposes a liftforce on a substance 18, indicated by arrow 20, and a drag force,indicated by arrow 22, as if the turbulence were not present. Inaddition, the turbulence applies forces which fluctuate rapidly in bothdirection and magnitude. Because these fluctuations are both rapid andrandom, it can be assumed that the fluctuating forces have an averagevalue of zero and have no effect other than to increase the apparentdiffusion coefficient of the macromolecules in the fluid.

[0035] As described herein, essentially all of the shear stress istransmitted by viscosity in the viscous sublayer but only part of it istransmitted by viscosity in the inertial sublayer, the rest beingtransmitted by turbulent fluctuations. Because of this, the lift forceapplied to a substance in the viscous sublayer, substance 1 in FIG. 4for example, can be substantially larger than that applied to asubstance of the same size in the inertial sublayer, such as substance 2in FIG. 4. This difference is far greater than would be accounted for ina laminar flow system by the difference in distance to the upstreamsurface of the permeable membrane from the locations of the twosubstances. To experience the large lift force generated in the viscoussublayer, the suspended substance, e.g., substance 1 in FIG. 4, ispreferably completely immersed in that sublayer (i.e., substances arepreferably smaller than the thickness of the viscous sublayer).Substances, e.g., substance 4, that are large compared to the thicknessof the viscous sublayer can still be small enough to experience thelesser lift force of the inertial sublayer, which can be a few orders ofmagnitude thicker than the viscous sublayer in most turbulent flows. Thelift force on a large substance, e.g., substance 3, which penetratespartially into the viscous sublayer but is so large that most of itssurface is in the inertial sublayer cannot be as large as that on asmall substance centered at the same location. From this a conclusioncan be drawn that the large lift force due to the large shear rate inthe viscous sublayer is available principally to substances that aresmall with respect to the thickness of that layer.

[0036] The permeate flow velocity is preferably small compared to theaverage tangential velocity in the fully turbulent region, e.g., lessthan one hundredth of the tangential velocity. In some embodiments, thepermeate velocity is on the order of about {fraction (1/1000)} of am/sec. or less while the average tangential velocity is on the order of10 m/sec or more. For example, for a 16P2 (16 inch or 40.64 cm rotordiameter) dynamic filtration machine manufactured by Pall Corporation,at 1800 rpm and 1000 lmh permeate rate, the average tangential velocitycan be about 19 m/sec, and the permeate velocity can be about 3×10⁻⁴m/sec. In this case, the permeate flow rate exerts a negligible effecton the thickness of the viscous sublayer.

[0037] In operation of embodiments of the invention, a substance (e.g.,a molecule of given size) can be separated or concentrated from asolution or suspension in which it is suspended by balancing the liftand drag forces exerted on it in the viscous sublayer by manipulatingthe shear rate and the permeate flow rate. For example, for a given sizesubstance at a given shear rate in the viscous sublayer, an upper limiton the permeate flow rate can be selected such that the permeate drag(e.g., as calculated by the Stokes formula) is equal to the lift. If thepermeate flow rate is increased above this point, the substance can bedragged through the permeable membrane. If the permeate flow rate is ator below this point, the substance can remain suspended on the upstreamside near the surface of the permeable membrane by the shear lift.Alternatively, or in addition, for a given size substance at a givenpermeate flow rate, an upper limit on the shear rate in the viscoussublayer can be selected such that the shear lift is equal to thepermeate drag. If the shear rate is decreased below this point, thesubstance can be dragged through the permeable membrane. If the shearrate is at or above this point, the substance can remain suspended onthe upstream side near the surface of the permeable membrane.

[0038] The permeate flow establishes the drag force acting on substancesupstream of the permeable membrane. In essence, the process fluid isdrawn through the membrane (i.e., from the upstream side of the membraneto the downstream side) into the permeate, which generally includessubstances smaller than the shear separation size. Any method ofcreating permeate flow can be employed in the context of the presentinvention. Generally, the permeate flow is established by creating adrop in pressure across the permeable membrane. The rate of permeateflow can be controlled in any number of ways, including, for example, byrestricting the flow of permeate in the permeate outlet of a separationdevice. The characteristics of the permeable membrane can also be usedto affect the permeate flow rate (e.g., thickness, voids volume, etc.).In addition, any means of adjusting the transmembrane pressure can beused to control the permeate flow rate.

[0039] The shear lift is also created in proximity to the surface of thepermeable membrane. Any method of creating shear within the processfluid can be employed in the context of the present invention.Typically, shear is established by effecting relative motion of theprocess fluid in a direction tangential to the surface of the permeablemembrane. The shear then establishes the lift force acting on particlesupstream of the permeable membrane. As discussed herein, the lift actingon some particles is sufficient to retain them upstream of the membrane,even in the presence of the permeate flow.

[0040] The shear separation size for a given protocol reflects thebalance of the drag force and lift force acting on the particlesupstream of the membrane. The opposition of these forces is illustratedin FIG. 3. In general, the drag force increases with increasing particlesize and with increasing permeate flow rate. Accordingly, for a givensize particle, the drag force can be varied by varying the permeate flowrate. The lift force which opposes the drag force increases withincreasing particle size, with increasing shear rate, and with adecreasing distance from the permeable membrane. Accordingly, for agiven size particle and a given distance from the permeable membrane,the lift force can be varied by varying the shear rate. The net forceacting on the particle can be adjusted to any value by suitablybalancing the shear rate and the permeate flow rate. Therefore, thebalance of lift and drag forces retards the transmembrane passage ofsubstances larger than the separation size yet allows substances smallerthan the separation size to pass through the permeable membrane into thepermeate. Utilizing the balance between lift force and drag force as theprimary means for establishing separation size enhances the precisionwith which substances above and below the cutoff size can be separated.Thus, in some embodiments, microfiltration or ultrafiltration can beachieved with more sharply tuned size and/or molecular weight cutoffcharacteristics.

[0041] A membrane for use in the shear separation systems, methods, anddevices can be of any type suitable for generating sufficient shear atthe membrane surface, and suitable porous membranes are known in theart. The membrane can be fashioned from any suitable material (e.g.,metal, ceramic, paper, polymer, etc.), so long as it is compatible withthe process fluid to be treated. Moreover, the dimensions of themembrane can also vary considerably within the boundaries ofcompatibility with the inventive systems and methods. Thus, the membranecan be of any suitable size or thickness. Moreover, as the shear isprimarily responsible for establishing the shear separation size,membranes for use in the present invention can have a wide variety ofpore ratings or molecular weight cutoffs. However, as discussed herein,the surface of the membrane is preferably sufficiently smooth toestablish a viscous sublayer adjacent to the surface. For the purpose ofestimating the effect on the viscous sublayer, at least two measures ofsmoothness are appropriate for a permeable membrane. Both the pore sizeand the variation of the height of the surface of the membrane betweenthe pores are preferably small compared to the thickness of the viscoussublayer. For the purpose of shear separation, a smooth membrane whichdoes not significantly interfere with the formation of the viscoussublayer can be, for example, one for which the sum of the pore size andthe root mean square variation of surface height is less than about onefifth of the thickness of the viscous sublayer.

[0042] Because the separation size is a function of the shear andpermeate flow, the pore rating of the membrane (or molecular weightcutoff) is not directly related to resolution. The permeable membrane isutilized primarily as a surface adjacent to which the shear flow isgenerated. Thus, one advantage of the present inventive method andsystem is that separation sizes smaller than the pore rating of themembrane can be achieved, thereby minimizing membrane fouling increasingtransmission, and prolonging the usable life of a separation system.This can also reduce the number of separation steps required, forexample, in the method of isolating a single protein molecule from acell lysate suspension or separating a protein from a milk product. Asecond advantage is that a broad range of separation cutoff sizes can beachieved with one membrane by varying the ratio of shear lift topermeate drag. This makes it possible to perform several separationsteps of a separation method with a single permeable membrane simply byrecycling the permeate of one step as the feed of the next step with theshear lift and permeate drag adjusted for a smaller or larger separationcutoff size. Therefore, the pore size or molecular weight cutoff of themembrane preferably is larger than the separation cutoff size, such asat least one and a half or at least twice the desired separation size(e.g., at least three times the separation size), and can be as much asat least five times the separation size (such as at least eight timesthe desired separation size) or even larger (e.g., at least ten timesthe desired separation size, or even up to about fifteen times thedesired separation size or larger). Thus, for example, where the desiredseparation size is less than about 70 kD, a membrane with a pore ratingof about 100 kD is suitable, although membranes with larger pores canalso be employed. Moreover, where the desired separation size is about100 kD to about 150 kD, a membrane with a pore rating of about 300 kD toabout 500 kD is suitable. Alternatively, the separation medium can havea pore size or a molecular weight cutoff substantially equal to thedesired separation cutoff size. In this embodiment, the permeablemembrane also serves as a “last chance” filter. Of course, the porerating of a membrane is preferably not smaller than the predeterminedshear separation cutoff size. Generally, the pore rating of a membranesuitable for shear separation has a pore rating of less than about 1000kD, as larger pores adversely impact the desired smoothness of themembrane surface. More preferably, the pore rating is about 500 kD orless (e.g., about 300 kD or less), or even about 100 kD or even smaller.However, a membrane having pores larger than even 1000 kD could beemployed in the inventive shear separation methods and systems if thesurface can be rendered sufficiently smooth to establish liftappropriate to balance the drag, as mentioned herein.

[0043] The inventive shear separation systems and methods provide meansof treating any suitable fluids. For example, the invention shearseparation systems and methods may be used to separate various proteinsfrom a wide variety of protein-containing liquids, including blood orblood fractions and cell cultures or lysates of cell cultures, such asyeast, bacteria, plant and mamalian cell cultures.

[0044] A particular example is the treatment of milk products,particularly skim milk and whey products. The present invention is wellsuited to treat any type of milk product, especially skim milk and wheyproducts. Skim milk is prepared by removing the cream from whole milk,and skim milk products include raw skim milk as well as fractionstherefrom (e.g., milk or milk products which have been previouslysubjected to filtration or other separatory methods, including but notlimited to whey and whey products). Whey typically is produced in cheesemaking, although the present invention is intended to encompass the useof other types of whey. Whey products include whey as well as fractionstherefrom. As such, whey products include, for example, cheese whey,clarified whey, whey powder, pasteurized whey, whey concentrates, andother whey fractions (e.g., whey or whey products which have beenpreviously subjected to filtration or other separatory methods).

[0045] If proteins are to be recovered from the milk product, it isdesirable that the milk product not be heated to a temperature whichcould adversely affect the proteins therein (e.g., denature theproteins, particularly the immunoglobulins or recombinant proteins,therein). In particular, the milk product is desirably maintained at atemperature of about 60° C. or less, preferably at a temperature ofabout 50° C. or less, more preferably at a temperature of about 40° C.or less, and most preferably at about 20-25° C. or below. Thus, the milkproduct is preferably neither pasteurized nor derived from a pasteurizedmilk product. Preferably, the milk product, such as raw skim milk orwhey, is obtained as a product from milk which has been subjected tofiltration, such as in accordance with the inventive method or viadynamic filtration such as disclosed in U.S. Pat. Nos. 5,256,437,5,356,651, and 5,401,523 to remove bacteria therefrom.

[0046] Thus, the present invention provides systems and methods oftreating a milk product, particularly the bacteria-depleted andfat-depleted milk product (most particularly such a raw skim milk orwhey-derived permeate), to concentrate a protein therein by treating themilk product via shear separation to form a protein-enriched retentateor a protein-enriched permeate (depending on the size of the proteinrelative to the separation size). Typically, a whey product containsmany proteins of different molecular weights, particularlyimmunoglobulins, lactoferrin, lactoperoxidase, blood serum albumin,β-lactoglobulin, α-lactalbumin, and/or any recombinant proteins. Othermilk products typically contain these same proteins, as well as casein.The present invention allows for the concentration of a particularprotein or combination of proteins (e.g., first, second, third, fourth,and fifth (as well as any other) proteins) by the selection ofparticular shear separation conditions (e.g. the pore rating of thefiltration medium, the drag force, and the lift force), and,alternatively, the use of a series of separation steps using differentprocessing conditions (e.g., filtration media having increasinglysmaller pore ratings, enhanced permeate flow, decreased lift, and evenconventional separation techniques in conjunction with the presentinventive method).

[0047] In the aforedescribed protein concentration method, the first,second, third, fourth, and fifth (as well as any other) proteins can beany suitable proteins. Desirably, proteins of higher molecular weight(MW) are concentrated before proteins of lower molecular weight.Typically, the proteins will be selected from the group consisting ofimmunoglobulins, lactoferrin, lactoperoxidase, blood serum albumin(usually bovine serum albumin when the ultimate source of the milkproduct is bovine milk), β-lactoglobulin, and α-lactalbumin. When themilk product contains all of these proteins, then, typically, the firstprotein will be immunoglobulins (generally about 150-900 kD MW); thesecond protein will be lactoferrin (generally about 74-90 kD MW) andlactoperoxidase (generally about 77.5 kD MW); the third protein will beblood serum albumin (generally about 66 kD MW); the fourth protein willbe β-lactoglobulin (generally present in dimeric form of about 36 kDMW), and the fifth protein will be α-lactalbumin (generally about 14 kDMW). The present inventive systems and methods, of course, areapplicable to other proteins, of larger or smaller molecular weight. Ofcourse, where the milk product also contains a recombinant protein, itsmolecular weight generally will be known. Similarly, the presentinventive systems and methods can be used to remove combinations ofproteins based on the molecular weights of the proteins relative to theseparation size of the separation protocol used. For example, instead ofseparately removing blood serum albumin and β-lactoglobulin from a milkproduct, these proteins can be separated together by a shear separationprotocol creating drag sufficient to draw the albumin andβ-lactoglobulin through the membrane, while creating lift sufficient toretain larger particles within the retentate. Of course, othercombinations of proteins can be separated in a similar manner.

[0048] If the initial milk product is conventional raw skim milk orwhey, then, after the aforesaid proteins are separated from the milkproduct, the resulting permeate will be fat- and protein-depleted andshould contain only minerals, vitamins, and lactose. The removal andconcentration of the various proteins can be accomplished in accordancewith the present invention by, for example, subjecting the milk productpermeate to the aforedescribed sequential shear separation protocolswherein (a) the first separation protocol effects a separation size ofabout 900 kD or more, preferably about 900-1,000 kD, to reject anyresidual fat and casein and to concentrate the proteins of interest inthe permeate (although, as described herein, this step preferably isomitted in some embodiments of the present invention), (b) the secondseparation protocol effects a separation size of about 90 kD or more,preferably about 90-100 kD, to form an immunoglobulin-enrichedretentate, (c) the third separation protocol effects a separation sizeof about 60-70 kD, preferably about 60-65 kD , to form alactoferrin/lactoperoxidase-enriched retentate, (d) the fourthseparation protocol effects a separation size of about 40-60 kD,preferably about 50-60 kD, to form a blood serum albumin-enrichedretentate, (e) the fifth separation protocol effects a separation sizeof about 15-25 kD, preferably about 15-20 kD, to form aβ-lactoglobulin-enriched retentate and an α-lactalbumin-enrichedpermeate, and/or (e) the sixth separation protocol effects a separationsize of about 10 kD or less, preferably about 5-10 kD, to form aβ-lactalbumin-enriched retentate and a substantially protein-depletedpermeate containing smaller molecular weight components, such aspeptides, minerals, vitamins, and lactose.

[0049] Any suitable device or system, such as a dynamic filtrationassembly, can be used for the generation of the shear forces and thepermeate flow. The dynamic filtration assembly can be of any suitableconfiguration and typically will include a housing containing aseparation assembly having at least one separation medium (preferablycontaining a permeable membrane) and a mechanism to effect relativemovement between the fluid being filtered and separation medium. Thehousing can include a process fluid inlet arranged to direct processfluid into the housing and a permeate outlet arranged to direct permeatefrom the housing. In addition, the housing also can include a retentateoutlet arranged to direct retentate from the housing.

[0050] Where the separation medium includes a permeable membrane, themembrane typically has an upstream surface which communicates with theprocess fluid inlet and a downstream surface which communicates with thepermeate outlet. Depending upon the desired shear separation protocol,the permeable membrane can have pores larger (even substantially larger,as described herein) than the size of substances to be retained upstreamof the permeable membrane. Moreover, to maximize the depth of theviscous sublayer, the permeable membrane can additionally have a surfaceroughness which is small compared to a viscous sublayer thickness on atleast its upstream surface, as described herein. To effect the shearseparation as herein described, the mechanism to effect relativemovement between the fluid being filtered and separation elementgenerally is preferably a means for generating a shear adjacent to theupstream surface of the permeable membrane. Such means preferablyestablishes shear forces in the process fluid sufficient to retard thepassage of substances to be retained upstream of the permeable membranein the presence of a permeate flow. The shear forces thus generated arepreferably sufficient to retard the passage of substances smaller thanthe viscous sublayer thickness through the permeable membrane in thepresence of the permeate flow.

[0051] The separation assembly and the mechanism to effect relativemovement between the fluid being filtered and separation medium can haveany of a variety of suitable configurations. For example, the separationassembly can comprise stationary filter elements and rotatinginterleaved discs or rotating filter elements and stationary interleaveddiscs such as those disclosed in International Publication No.WO95/00231 and International Publication No. WO96/01676. Alternatively,the separation assembly can comprise stationary filter elements and arotatable housing such as those described in International PublicationNo. WO97/13571.

[0052] Process fluid which is to undergo shear separation can besupplied to a dynamic filtration assembly by any number of means, forexample, by any of the means described in International Publication Nos.WO95/00231 and WO96/01676. The process fluid can be directed through theprocess fluid inlet into the housing, for example, at a predeterminedpressure and flow rate. The process fluid then flows tangentially alongthe upstream surface of the permeable membrane and out of the housingthrough the retentate outlet. The transmembrane pressure forces permeatethrough the permeable membrane from the upstream surface to thedownstream surface, and the permeate is removed from the housing via thepermeate outlet.

[0053] As the process fluid is introduced into the housing of thedynamic filtration assembly, an angular momentum is imparted to theprocess fluid causing it to rotate at a particular velocity. Therotation of the fluid effects the shear described herein. The angularmomentum can be imparted to the fluid in any number of ways. Forexample, as described in International Publication No. WO96/01676,relative rotation between the separation assembly and a separate memberfacing the separation medium can be produced, e.g., the member can berotated or the separation medium can be rotated. Alternatively, asdescribed in International Publication No. WO97/13571, the housing canbe rotated. In both cases, the rotational movement transfers an angularmomentum to the process fluid thereby creating a shear. As describedherein, the shear generates lift on particles or substances in theprocess fluid while permeate flow generates drag on particles orsubstances in the process fluid. Both of these parameters can beadjusted to generally balance the lift and drag forces on a particle orsubstance at a predetermined separation size as described herein. Forexample, the shear rate can be adjusted by changing the revolutionvelocity, and the permeate flow can be adjusted by changing thetransmembrane pressure.

[0054] A shear separation system embodying the invention may include acontrol system and a dynamic filtration assembly. As discussed above, ashear separation system may be used to separate particles or substancesof varying size, even when a separation medium has a separation cutoffsize greater than the size of the particles of interest. The effectiveseparation cutoff size of a dynamic filter assembly depends principallyon shear lift and permeate drag forces. More particularly, the effectiveseparation cutoff size can be controlled by adjusting the relativemagnitudes of shear lift and permeate drag forces. Accordingly, byutilizing a control system which senses the effective separation cutoffsize and varies shear lift and/or permeate drag, the effectiveseparation cutoff size of a dynamic filter assembly can be controlledaccording to the needs of a particular application.

[0055] An exemplary embodiment of a shear separation system including acontrol system 30 and a dynamic filter assembly 36 coupled to thecontrol system 30 is illustrated in FIG. 5. The control system 30comprises a sensor 32 and a controller 34 coupled to the sensor 32. Boththe controller 34 and the sensor 32 may also be coupled to the shearseparation device 36.

[0056] The dynamic filter assembly 36 comprises any device capable ofseparating particles of varying size using shear lift and permeate dragforces to effect separation. For example, the dynamic filter assembly 36may create shear lift by rotating either a housing, one or moreseparation elements, or one or more disks or cylinders in contact with aprocess fluid. The shear separation system preferably also includes anarrangement of pumps, valves, and conduits to control fluid flow throughthe housing. For example, the dynamic filter assembly may include ahousing suitable for containing the process fluid. One or moreseparation elements may divide the housing into a process fluid regionand a permeate region. The housing may include a process fluid inlet todirect process fluid into the process fluid region of the housing and aretentate outlet to direct retentate fluid from the process fluid regionof the housing. The housing may also include a permeate outlet to directpermeate fluid from the permeate region of the housing. A pump may belocated upstream or downstream of the housing to control a fluid flowrate. The dynamic filter assembly may include a motor to generate shearlift by rotating the housing, the separation elements, and/or the disksor cylinders.

[0057] An exemplary dynamic filter assembly is disclosed inInternational Publication Number WO 97/13571, published on Apr. 17,1997. However, the present invention is not limited to any particulartype of dynamic filter assembly. Any device which effects separationusing shear lift and permeate drag forces is within the scope of theinvention.

[0058] The sensor 32 may comprise any type of sensor capable of beingcoupled to a dynamic filter assembly to measure the performance of theassembly. For example, the sensor 32 may comprise a particle size sensorwhich produces a signal, e.g., a current or a voltage, indicative of thesize of one or more of the particles or substances in the permeate orthe retentate and, therefore, indicative of the measured separationcutoff size of the dynamic filter assembly. In a preferred embodiment,the sensor 32 is coupled to a permeate conduit downstream of aseparation medium in the dynamic filter assembly 36 to sense the size ofparticles or substances in the permeate and produce a signal indicativeof the measured separation cutoff size of the device. Alternatively, thesensor 32 may be coupled to a retentate conduit on an upstream side ofthe separation medium to sense the size of particles in the retentate. Avariety of suitable sensors 32, including counters or turbidity sensors,are readily available.

[0059] The controller 34 comprises any type of controller suitable forproviding an output signal to vary shear lift and/or permeate drag inthe dynamic filter assembly 36 based on one or more inputs. Thecontroller 34 preferably receives a plurality of input signals andproduces at least one output signal. In the illustrated embodiment, thecontroller 34 may receive a reference input signal indicative of adesired separation cutoff size and another input signal indicative of ameasured or sensed separation cutoff size, e.g., the output from thesensor 32. The controller 34 preferably produces an error signalindicative of the difference between the reference input signal and theoutput from the sensor 32, and the error signal may comprise the outputsignal of the controller 34. In a preferred embodiment, the controller34 processes the error signal into a format suitable for controllingoperating parameters, e.g., parameters, such as rotational velocityand/or transmembrane pressure, which affect shear lift and/or permeatedrag, in the dynamic filter assembly 36.

[0060] The controller 34 may be analog or digital and may includeappropriate conditioning circuitry to condition the input signals. Forexample, if the inputs are analog signals, and the controller 34 isdigital, the controller 34 may include an analog-to-digital (A/D)converter to convert the input signals into digital format for furtherprocessing. The controller 34 may also include a processing circuit,e.g. a microprocessor, which implements a control law and outputs acontrol signal to the dynamic filter assembly 36. The controller 34 mayalso include a digital-to-analog (D/A) converter to convert the controlsignal into an analog format for use in an analog device, e.g., themotor which drives the housing, filter elements, or disks of the dynamicfilter assembly 36. In a preferred embodiment, the controller comprisesa programmable logic controller (PLC).

[0061] In operation, a process fluid flow is established through thedynamic filter assembly 36, creating a transmembrane pressure across theseparation medium in the device. The transmembrane pressure creates apermeate fluid flow which, in turn, creates a permeate drag force onparticles in the process fluid on an upstream side of the separationmedium. A shear lift force is produced on particles in the fluid on theupstream side of the separation medium, for example, by actuating amotor to rotate the housing, the filter elements, or the disks orcylinders in contact wit the process fluid. The shear lift force opposesthe permeate drag force on the particles on the upstream side of theseparation medium. By adjusting parameters affecting one or both ofthese forces, substances of a first size may remain on the upstream sideof a separation medium and substances of a second size may pass throughthe separation medium to the downstream side. In a preferred embodiment,the sensor 32 senses the size of substances in the permeate or retentateand produces an output signal indicative of the measured separationcutoff size of the dynamic filter assembly 36.

[0062] The controller 34 receives one or more output signals from thesensor 32 and also receives one or more reference input signalsindicative of a desired system output, e.g., a desired separation cutoffsize for the dynamic filter assembly 36. In response, the controller 34outputs one or more control signals to the dynamic filter assembly 36.For example, if the relationship between the sensor output and thereference input is within a predetermined tolerance, indicating, forexample, that the measured separation cutoff size is equal orsufficiently close to the desired separation cutoff size, the controller34 may output a control signal which does not significantly affect theoperation of the dynamic filter assembly 36 because the assembly isoperating as desired. If the relationship between the sensor output andthe reference input is outside the predetermined tolerance, thecontroller 34 may output a control signal which affects the generationof dynamic filter assembly 36 in a manner which forces the relationshipback within the predetermined tolerance. The controller 34 may increaseor decrease the shear lift on the substances in the process fluid; thecontroller 34 may increase or decrease the permeate drag on thesubstances in the process fluid; or the controller 34 may increase ordecrease both the shear lift and the permeate drag on the substances.

[0063] The mechanism by which the controller 34 causes the dynamicfilter assembly 36 to adjust the shear lift and/or permeate drag dependson the type of dynamic filter assembly 36 being used to effect shearseparation. For example, if the dynamic filter assembly utilizesrelative rotation between the process fluid and the separation elementsto create shear lift, the controller 34 may vary the speed of the motorwhich causes the rotation, thereby increasing or decreasing the shearlift. Alternatively, or additionally, the controller 34 may control oneor more pumps to adjust the pressure differential across the separationelements in the dynamic filter assembly to increase or decrease thepermeate flow rate and thereby adjust the permeate drag. The presentinvention is not limited to any particular mechanism for adjusting shearlift and/or permeate drag. Any method for controlling the relativemagnitudes of shear lift and permeate drag are within the scope of theinvention.

[0064] Although the illustrated embodiment depicts a single sensor 32, asingle controller 34, and a single feedback loop, the present inventionis not limited to such an embodiment. A control system may include aplurality of sensors which sense a plurality of outputs from aseparation device. For example, sensors which sense the speed and/oracceleration of the motor may be included in addition to sensors whichsense the size of the particles. Thus, the controller 34 may receiveinputs from a plurality of sensors, implement a plurality of controllaws and output a plurality of control signals to the dynamic filterassembly.

[0065] A shear separation method and system of the present invention canbe used in a single pass mode or in a recirculation mode. For example,the retentate drawn through the retentate outlet can be redirected tothe process fluid inlet for additional separation. Alternatively or inaddition to the recirculation of the retentate, the permeate can berecirculated by drawing the permeate from the permeate outlet and intothe process fluid inlet.

[0066] In addition to complete separation or substantially completeseparation, the shear separation systems and methods of the presentinvention may also be utilized for concentration of the substanceshaving a size less than the separation cutoff size and/or the substanceshaving a size greater than the separation cutoff size. For example,substances having a size less than the predetermined separation cutoffsize can pass through the permeable membrane in the permeate flow,resulting in a higher concentration of those substances on thedownstream side of the permeable membrane than on the upstream side.Similarly, substances having a size greater than the predeterminedseparation cutoff size can be retained along the upstream side of thepermeable membrane, resulting in a higher concentration of thosesubstances on the upstream side of the permeable membrane than on thedownstream side. Accordingly, the permeate can comprise a higherconcentration of substances less than the predetermined separationcutoff size and the retentate can comprise a higher concentration ofsubstances greater than the predetermined separation cutoff size.

EXAMPLES

[0067] The following examples further illustrate the present invention.In particular, they demonstrate that the inventive shear separationsystems and methods can effect a separation size substantially smallerthan the pore rating of any separation medium employed. The examplestherefore demonstrate that the balance of shear lift and permeate dragforces can achieve separation of particles from a solution or suspensionsubstantially independently of the size exclusion of the membrane. Theexamples further demonstrate that the inventive shear separation methodcan be employed to recover proteins from milk products. Of course, asthese examples are included for illustrative purposes, they should notin any way be construed as limiting the scope of the present invention.

Example 1

[0068] This example demonstrates that the balance of shear lift andpermeate drag forces can achieve separation of particles from a solutionor suspension regardless of the size exclusion of the membrane.

[0069] In this example, the process fluid comprised a phosphate bufferedsaline solution (120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer) with a0.1% concentration of Bovine Serum Albumin (BSA), which is approximately60 kD in size, and a 0.02% concentration of Lysozyme, which isapproximately 16 kD in size. High Pressure Liquid Chromatography (HPLC)was used to determine the protein concentrations during the experiment.The tests were conducted at several spin rates (different shear rates)and permeate flow rates. A polyethersulfone membrane available from PallCorporation under the trade designation FILTRON OMEGA UF was used in twodifferent pore sizes in a Pall Corporation LAB6 dynamic filtrationdevice. One pore size was such as to exclude proteins larger than 300kD, and the other was such as to exclude proteins larger than 70 kD.

[0070]FIG. 6A is a graph illustrating the results of two tests (test 1and test 2) utilizing the 300 kD Filtron Omega UF membrane. The permeateduct of the grid on which the membrane was supported was restricted sothat a permeate flow rate of approximately 30 ml/min was obtained. Theretentate flow rate was about 1 l/min. For both tests, permeate wascollected first at 3450 rpm, then at 3000, 2500, 2000, 1500, and 900 rpmin that order. These spin rates produce shear at the membrane surface of5×10⁵ sec⁻¹, 3×10⁵ sec⁻¹, 2.5×10⁵ sec⁻¹, 1.75×10⁵ sec⁻¹, 1.0×10⁵ sec⁻¹,and 4×10⁴ sec⁻¹, respectively. A second sample was then collected at aspin rate of 3450 rpm. In the second test (test 2), an additional samplewas taken at 2000 rpm with the retentate rate reduced to 0.5 l/min. Atthe end of the first test, a portion of the membrane, approximately 0.5cm across, was found to have been torn off the support material.However, a dye test of the membrane had shown good uniformity of proteindeposit over the grooves, which indicates reasonably uniform flow inspite of the tear. In the second test, the membrane was in goodcondition at the end of the experiment.

[0071] The pore structure of the 300 kD membrane is much too large torestrict transmission of either BSA or Lysozyme. This is shown in FIG.6A by the 100% transmission of these two proteins at zero spin rate. Upto 1500 rpm, there is a distinct reduction in protein transmissionthrough the membrane with increasing spin rate, with the heavier BSAbeing more strongly affected than the Lysozyme in each test. At higherspin rates, there is an indication that transmission can increase withspin rates. This is possibly a result of reducing the viscous sublayerthickness to the point that the surface roughness of the 300 kD media issufficient to disrupt the viscous sublayer. The shift in the second 3450point can be due to fouling of the membrane, or it can simply be anindication of scatter in the data. The shift in the second 2000 rpmpoint could be for the same reason. It is not large enough to concludethat it is due to retentate flow rate reduction. These experimentsdemonstrate the separation of proteins of two distinct sizes by abalance of shear lift and permeate drag forces alone, i.e., without anyreliance on the size exclusion of the membrane.

[0072]FIG. 6B is a graph illustrating the results of the tests (test 1and test 2) utilizing the 70 kD Filtron Omega UF membrane. The secondtest utilizing the 70 kD membrane was conducted in the same manner asthe two experiments with the 300 kD membranes except that the permeateflow rate was 18 ml/min, and the reduced retentate point at 2000 rpm wasnot tested. In the first test, the permeate rate was set at 160 ml/min,and a new membrane was used for each data point.

[0073] As illustrated in the graph, transmission of BSA through the 70kD membrane is highly restricted, so interpretation of this graph iscomplicated by the likelihood of a BSA gel layer on the membrane. It issuspected that the 160 ml/min permeate flow rate is a large enoughpermeate rate to drag most of the Lysozyme through the shear layer atall spin rates, while 18 ml/min is so slow that the Lysozyme cannotpenetrate the BSA gel layer. These last two tests demonstrate the use ofshear forces balanced against drag forces to separate small proteinswhile large proteins are excluded by conventional filtration.

Example 2

[0074] This example demonstrates that the inventive shear separationmethod can produce an effective separation size substantially smallerthan the pore rating of the filter medium employed.

[0075] In particular, an attempt was made to separate proteins from asuspension of lysed E. coli cells in PBS using an experimentalpolyethersulfone membrane from Pall Corporation called PV-20. The PV-20is a double skinned polyethersulfone membrane that rates as anapproximately 100 kD molecular weight cutoff. Electrophoresis gels ofthe permeate were used to determine protein concentration. In theexperiment, permeate flow was set to 13 ml/min, and a rotation rate of1935 rpm was used which produces a shear rate at the membrane surface onthe order of 10⁵ sec⁻¹. The table below summarizes the results of theexperiment. Concentration Protein Size Ratio at Concentration (kD) 1935rpm Ratio at 0 rpm 170 0.00% 0.00% 119 0.00% 54.83% 95/86 (two bands)0.00% 70.55%  41 16.74% 96.91%  27 37.02% 105.57%

[0076] As indicated by the results in the table, the transmission ofproteins was significantly impeded by the shear even though the pores ofthe membrane were large enough to allow transmission of much largermolecules.

Example 3

[0077] This example illustrates how the shear separation method can beemployed to recover the proteins from milk.

[0078] Whole milk is subjected to centrifugal separation or dynamicfiltration (using a filtration medium having a pore rating of 0.8 μm) toproduce a cream retentate and a skim milk permeate. The skim milkpermeate is dynamically filtered (using a filtration medium having apore rating of 0.4 μm) to produce a fat-enriched retentate and afat/bacteria-depleted permeate.

[0079] The fat/bacteria-depleted permeate is subjected to shearseparation using a filtration medium having a pore rating of 0.3 μm togenerate an effective separation size of 0.05-0.2 μm to produce amicellar casein-enriched retentate (having a 15-20% total solidsconcentration, with a 60-70% micellar casein concentration of the totalsolids) and a milk serum permeate.

[0080] The milk serum permeate is subjected to a shear separation stepusing a filtration medium having a molecular weight cutoff of 500 kD togenerate an effective separation size of 100 kD to produce animmunoglobulin-enriched retentate and an immunoglobulin-depleted milkserum permeate. The immunoglobulin-depleted milk serum permeate issubjected to shear separation using a filtration medium having amolecular weight cutoff of 200 kD to generate an effective separationsize of 70 kD to produce a lactoferrin/lactoperoxidase-enrichedretentate and a lactoferrin/lactoperoxidase-depleted milk serumpermeate. The lactoferrin/lactoperoxidase-depleted milk serum permeateis recirculated through the filter and subjected to shear separationunder conditions of decreased lift and/or increased drag to generate aneffective separation size of 50 kD to produce a bovine serumalbumin-enriched retentate and animmunoglobulin/lactoferrin/lactoperoxidase/bovine serum albumin-depletedpermeate. Alternatively, the initial milk serum permeate can besubjected to an initial shear separation protocol using a filtrationmedium having a molecular weight cutoff of 200 kD with a suitablelift/drag ratio to generate an effective separation size of 50 kD toproduce an immunoglobulin/lactoferrin/lactoperoxidase/bovine serumalbumin-depleted permeate.

[0081] The immunoglobulin/lactoferrin/lactoperoxidase/bovine serumalbumin-depleted permeate is recirculated through a 100 kD filter andsubjected again to shear separation under conditions of decreased liftand/or increased permeate flow to generate an effective separation sizeof 20 kD to produce a β-lactoglobulin-enriched retentate and aβ-lactoglobulin-depleted permeate. The β-lactoglobulin-depleted permeateis recirculated through the same filter and subjected again to shearseparation under conditions of decreased lift and/or increased permeateflow to generate an effective separation size of 10 kD to produce anα-lactalbumin-enriched retentate and an α-lactalbumin-depleted permeate(containing non-fat, non-protein milk solids).

Example 4

[0082] This example illustrates how the shear separation method can beemployed to recover the proteins from whey.

[0083] Cheese whey is subjected to centrifugal separation or dynamicfiltration (employing a shear separation protocol generating aneffective separation size of 0.65 μm) to produce a whey cream retentateand a fat-depleted whey permeate. The fat-depleted whey permeate isdynamically filtered (using a filtration medium having a pore rating of0.4 μm) to produce a fat-enriched retentate and a fat/bacteria-depleted,protein-enriched permeate. The fat/bacteria-depleted, protein-enrichedpermeate is dynamically filtered (using a filtration medium having apore rating of 0.2 μm) to produce fat/bacteria-enriched sludge retentateand a whey protein-enriched permeate.

[0084] The whey protein-enriched permeate is subjected to a shearseparation step using a filtration medium having a molecular weightcutoff of 500 kD to generate an effective separation size of 100 kD toproduce an immunoglobulin-enriched retentate and animmunoglobulin-depleted whey product permeate. Theimmunoglobulin-depleted whey product permeate is subjected to shearseparation using a filtration medium having a molecular weight cutoff of200 kD to generate an effective separation size of 70 kD to produce alactoferrin/lactoperoxidase-enriched retentate and alactoferrin/lactoperoxidase-depleted whey product permeate. Thelactoferrin/lactoperoxidase-depleted whey product permeate isrecirculated through the filter and subjected to shear separation underconditions of decreased lift and/or increased drag to generate aneffective separation size of 50 kD to produce a bovine serumalbumin-enriched retentate and animmunoglobulin/lactoferrin/lactoperoxidase/bovine serum albumin-depletedpermeate. Alternatively, the initial whey product permeate can besubjected to an initial shear separation protocol using a filtrationmedium having a molecular weight cutoff of 200 kD with a suitablelift/drag ratio to generate an effective separation size of 50 kD toproduce an immunoglobulin/lactoferrin/lactoperoxidase/bovine serumalbumin-depleted permeate.

[0085] The immunoglobulin/lactoferrin/lactoperoxidase/bovine serumalbumin-depleted permeate is recirculated through a 100 kD filter andsubjected again to shear separation under conditions of decreased liftand/or increased permeate flow to generate an effective separation sizeof 20 kD to produce a β-lactoglobulin-enriched retentate and aβ-lactoglobulin-depleted permeate. The β-lactoglobulin-depleted permeateis recirculated through the same filter and subjected again to shearseparation under conditions of decreased lift and/or increased permeateflow to generate an effective separation size of 10 kD to produce anα-lactalbumin-enriched retentate and an α-lactalbumin-depleted permeate(containing non-fat, non-protein milk solids).

Example 5

[0086] This example demonstrates the concentration of a recombinantprotein from the milk of a transgenic mammal. In particular, a singlefilter is employed to separate a recombinant α-antitrypsin (αAT), aprotein of about 51 kD, from the milk. See Archibald et al, Proc. Nat.Acad. Sci. USA, 87(13), 5178-82 (1990).

[0087] Raw milk containing αAT obtained from a transgenic mammal isfirst processed to produce a milk serum (i.e., milk substantially freeof fat, bacteria, and micellar casein). The milk serum containing theαAT is dynamically filtered in accordance with the present inventionusing a membrane with a molecular weight cutoff of about 100 kD. Thefirst filtration step employs conditions of suitably high shear rate andlow permeate rate to draw most of the particles smaller than αAT (e.g.,the first permeate comprises β-lactoglobulin (about 35 kD in dimericform) and α-lactalbumin (about 15 kD)) through the membrane whileretaining αAT and larger particles in the retentate upstream of themembrane. The permeate is collected in a permeate collection chamber,and the retentate is collected in a retentate collection chamber.Thereafter, the retentate is again dynamically filtered in accordancewith the present inventive method using the same membrane. The lift onthe particles during the second filtration is suitably reduced (e.g., byslightly reducing the shear rate and/or increasing the permeate rate) soas to draw most of the αAT through the membrane yet maintained suitablyhigh enough to retain particles larger than the αAT (e.g.,immunoglobulins (about 150-900 kD), lactoferrin and lactoperoxidase(each about 75 kD), and blood serum albumin (about 60 kD)) in theretentate upstream of the membrane. The second permeate contains asubstantially greater concentration of αAT and a substantially reducedconcentration of other milk components than the initial process fluid,and is collected in a permeate collection chamber other than thatcontaining the first permeate.

Example 6

[0088] This example demonstrates that a smooth filter medium creates astronger, less turbulent shear boundary layer for filtration (allowingfor a longer filtration life). In particular, the effect of placing thesmooth side (i.e., the cast side) of an unsupported membrane facingupstream in contact with the process fluid, was evaluated as compared toplacing the rough side (i.e., the side opposite the cast side) of thesame unsupported membrane facing upstream in contact with the processfluid.

[0089] Each of two unsupported, 0.8 μm pore rated, nylon membranes fromthe same batch or roll of nylon membrane was mounted in a PallCorporation LAB6 dynamic filtration machine, with the only differencebeing the orientation of the membrane, namely either smooth or roughside facing upstream. Each of these filtration machines was fed milkfrom a common feed tank and a common pump. A flow decay test was run onboth machines in parallel. In this test, the permeate rate was broughtup to 400 ml/min, and the spin rate was brought up to 2100 rpm during ashort start up period. The feed pressure and valve settings weremaintained approximately constant on both machines, and the permeateflow was allowed to decay by whatever fouling occurred. The table belowsummarizes the results of the experiment. Smooth Side Rough SideUpstream - Upstream - Permeate Flow Permeate Flow Time (minutes)(ml/min) (ml/min) 2 0 0 6 128 130 10 400 12 394 15 404 16 420 30 413 37347 388 48 293 58 380 241 67 209 68 374 73 380 182 90 362 146 105 351 112120 348 86 135 323 60 150 315 46 165 310 38 180 302 28 195 300 26 210286 20 225 287 18 240 278 18 250 260 14

[0090] As indicated by the results in the table, the permeate flowdecayed much more significantly and quickly when the rough side of themembrane was facing upstream than when the smooth side of the membranewas facing upstream. In particular, within 58 min (about 1 hr), thepermeate flow was only 60% of the maximum observed permeate flow inconnection with the rough side of the membrane facing upstream, ascompared with a permeate flow of 90% of the maximum observed permeateflow in connection with the smooth side of the membrane facing upstream.Similarly, after 2 hr, 3 hr, and 4 hr, the permeate flow decreased to21%, 7%, and 4% of the maximum observed permeate flow, respectively, inconnection with the rough side of the membrane facing upstream, ascompared with permeate flows of 83%, 72%, and 66%, of the maximumobserved permeate flow, respectively, in connection with the smooth sideof the membrane facing upstream. These data demonstrate that a smoothfilter medium promotes a stronger, less turbulent shear boundary layerfor filtration.

[0091] All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

[0092] While this invention has been described with an emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that variations of the preferred embodiments may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the following claims.

What is claimed is:
 1. A method of treating a process fluid toconcentrate or separate substances having a size less than apredetermined separation size and substances having a size greater thanthe predetermined separation size comprising: generating a permeate flowthrough a permeable membrane; and creating a shear flow at the surfaceof the permeable membrane which retards passage through the permeablemembrane of substances having a size greater than the predeterminedseparation size and which allows passage through the permeable membraneof substances having a size less than the predetermined separation size.2. The method of claim 1, wherein generating a permeate flow through apermeable membrane includes generating a permeate flow through apermeable membrane having a pore size or molecular weight cutoff whichis larger than said separation size.
 3. The method of claim 2, whereinsaid pore size or molecular weight cutoff of said membrane is at leasttwice said separation size.
 4. The method of claim 3, wherein said poresize or molecular weight cutoff of said membrane is at least three timessaid separation size.
 5. The method of claim 4, wherein said pore sizeor molecular weight cutoff of said membrane is at least five times saidseparation size.
 6. The method of claim 5, wherein said pore size ormolecular weight cutoff of said membrane is at least eight times saidseparation size.
 7. The method of any of claims 1-6, wherein creating ashear flow includes establishing a viscous sublayer adjacent to anupstream surface of said permeable membrane.
 8. The method of claim 7,wherein creating a shear flow establishes a lift force which retards thepassage of particles smaller than the viscous sublayer thickness throughthe permeable membrane.
 9. The method of any of claims 1-8, wherein saidmembrane has a surface roughness small compared to the thickness of saidviscous sublayer.
 10. A shear separation method comprising: generating apermeate flow through a permeable membrane having a pore size ormolecular weight cutoff substantially larger than a predeterminedseparation size; and creating a shear flow adjacent to the upstreamsurface of the permeable membrane, including establishing shear forcesin a process fluid to retard the passage of substances to be retained onthe upstream surface of the permeable membrane.
 11. A shear separationmethod comprising: generating a permeate flow through a smooth permeablemembrane; and creating a shear flow boundary layer having a viscoussublayer adjacent to the upstream surface of the permeable membrane,including establishing sear forces in a process fluid to retard thepassage of particles smaller than the viscous sublayer thickness throughthe permeable membrane.
 12. A method of concentration comprising:generating a permeate flow through a permeable membrane from an upstreamsurface to a downstream surface; and creating a shear flow of a processfluid at the upstream surface of the permeable membrane which retardspassage through the permeable membrane of substances having a sizegreater than a predetermined separation size and which allows passagethrough the permeable membrane of substances having a size less than thepredetermined separation size, thereby concentrating at least one of:(i) the substances having a size greater than the predeterminedseparation size at the upstream surface of the permeable membrane, and(ii) the substances having a size less than the predetermined separationsize at the downstream surface of the permeable membrane.
 13. The methodof any of claims 1-12, wherein said process fluid is derived from milk.14. The method of any of claims 1-13, wherein said substance includes aprotein.
 15. The method of claim 14, wherein said protein includes arecombinant human protein.
 16. The method of claim 14 or 15, whereinsaid protein includes an immunoglobulin.
 17. A shear separation systemcomprising: a housing; a process fluid inlet arranged to direct processfluid into the housing; a permeate outlet arranged to direct permeatefrom the housing; at least one separation element disposed within thehousing and including a permeable membrane having an upstream surfacewhich communicates with the process fluid inlet and a downstream surfacewhich communicates with the permeate outlet, the permeable membranehaving a pore size or a molecular weight cutoff substantially largerthan the size of substances to be retained upstream of the permeablemembrane; and the separation element and the process fluid beingarranged to rotate relative to one another to generate a shear flowadjacent to the upstream surface of the permeable membrane and establishshear forces in the process fluid to retard the passage of thesubstances to be retained upstream of the permeable membrane.
 18. Ashear separation system comprising: a housing; a process fluid inletarranged to direct process fluid into the housing; a permeate outletarranged to direct permeate from the housing; at least one separationelement disposed within the housing and including a permeable membranehaving an upstream surface which communicates with the process fluidinlet and a downstream surface which communicates with the permeateoutlet; and the separation element and the process fluid being arrangedto rotate relative to one another to create a shear flow boundary layerhaving a viscous sublayer adjacent to the upstream surface of thepermeable membrane and establish shear forces in a process fluid toretard the passage of particles smaller than the viscous sublayerthickness through the permeable membrane, the permeable membrane havinga surface roughness small compared to a viscous sublayer thickness onthe upstream surface thereof.
 19. The shear separation system of claim17 or 18, further comprising at least one member disposed within thehousing facing the at least one separation element, the member and theseparation element being arranged to rotate relative to one another. 20.The shear separation system of claim 17 or 18, wherein the separationelement comprises a stationary separation element, and the shearseparation system further comprises a rotatable wall mounted around thestationary separation element and defining an axis of rotation, whereinthe upstream surface of the permeable membrane is generallyperpendicular to the axis of rotation and wherein the rotatable wall isarranged to rotate the process fluid across the upstream surface of thepermeable membrane.
 21. A method for controlling a shear separationsystem comprising: generating a permeate drag on substances in a processfluid; generating a shear lift on the substances in the process fluid;sensing a parameter indicative of the size of the substances; andadjusting at least one of the shear lift and the permeate drag based onthe sensed parameter.
 22. A shear separation system comprising: adynamic filter assembly capable of separating substances of varying sizein accordance with shear lift and permeate drag forces on thesubstances; a sensor coupled to the dynamic filter assembly to producean output signal; and a controller coupled to the sensor and the dynamicfilter assembly to adjust at least one of the shear lift and thepermeate drag forces in accordance with the output signal from thesensor.